Although not limited thereto, the invention especially applies to Stimulated Emission Depletion (STED) microscopy (S. W. Hell and J. Wichmann, Optics Letters 19, 780 (1994); T. A. Klar and S. W. Hell, Optics Letters 24, 954 (1999)) which is an ideal method for imaging with high spatial and temporal resolution. A resolution better than 6 nm (E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, Nature Photonics 3, 144 (2009)) can be reached. STED microscopy allows dynamic imaging (V. Westphal, M. A. Lauterbach, A. Di Nicola, and S. W. Hell, New Journal of Physics 9, 435 (2007)) with up to 200 frames per second (M. A. Lauterbach, C. Ullal, V. Westphal, and S. W. Hell, Langmuir 26, 14400 (2010)) and is compatible with imaging dynamics in living cells (V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, and S. W. Hell, Science 320, 246 (2008); B. Hein, K. I. Willig, and S. W. Hell, Proceedings of the National Academy of Sciences of the United States of America 105, 14271 (2008)) and even in tissue (U. V. Nägerl, K. I. Willig, B. Hein, S. W. Hell, and T. Bonhoeffer, Proceedings of the National Academy of Sciences of the United States of America 105, 18982 (2008); N. T. Urban, K. I. Willig, S. W. Hell, and U. V. Nägerl, Biophysical Journal 101, 1277 (2011)).
A common implementation of STED microscope is as a laser scanning microscope. The sample to image is provided with a substance, such as a fluorescent marker or fluorophores, having a first state with first spectral properties and a second state with second spectral properties. In particular, the fluorophores emit fluorescence when relaxing from the second state to the first state. In STED microscopy, an excitation focus is provided by a focused laser probing light beam illuminating the sample through an objective-lens assembly. The fluorescence ability of the fluorophores in an outer part of the excitation focus is transiently turned off. The switching of the molecules into a non-fluorescent state is achieved via stimulated emission with a laser transfer light beam (“STED beam”). The STED beam is commonly passed through a spiral phase mask of charge one, sometimes imaged with a 4f configuration onto a back focal plane of an objective-lens assembly, resulting in a toroidal focus having a first intensity area with a first intensity adapted to deplete the fluorophores in the second state to the first state, and a second intensity area with a second intensity adapted not to transfer the fluorophores between the first and second states (V. Westphal, M. A. Lauterbach, A. Di Nicola, and S. W. Hell, New Journal of Physics 9, 435 (2007)). Besides, in STED microscopy, fluorescence can be elicited in the stained sample by the probing light beam overlaid onto the STED focus of toroidal (“donut”) shape, at least partly onto the second intensity area. Thus, the ability of the fluorophores to emit is turned off via stimulated emission in the first intensity area corresponding to the periphery of the excitation focus. Only in the second area, at the very center, where the STED focus has close-to-zero intensity, the fluorophores are able to spend significant time in the fluorescent state. The second intensity area wherein the fluorophores are not switched off, and accordingly the minimally resolvable distance, shrink to zero with increasing intensity of the STED beam (S. W. Hell, Science 316, 1153 (2007)). The fluorescence of the fluorophores in the second area of the transfer light beam forms an optical measurement signal detected point by point by a probe detector.
It is, however, often desirable to locate and image structures of interest in a biological specimen as sample, such as cells, which do not strongly absorb light and do not fluoresce. Such structure of interest can hardly be located and imaged using STED microscopy even with the implementation of a second high-resolution color channel.
The invention aims to solve the above mentioned problem.
To that end, the invention proposes a microscope for high spatial resolution imaging a structure of interest in a sample comprising a substance, said substance having a first state with first spectral properties and a second state with second spectral properties, the microscope comprising:                an objective-lens assembly having first and second sides opposite to each other, and presenting first and second focal planes respectively on said first and second sides, the sample being intended to be placed on the first side,        a transfer light source arranged on the second side of the objective-lens assembly to emit a transfer light beam towards the sample through the objective-lens assembly, said transfer light beam having an intensity and a phase profile,        a light beam modulating device placed between the transfer light source and the objective-lens assembly, said light beam modulating device being adapted to spatially vary the transfer light beam so that said transfer light beam having passed trough said light beam modulating device and the objective-lens assembly presents at least a first intensity area with a first intensity adapted to transfer the substance in the second state to the first state, and at least a second intensity area with a second intensity adapted not to transfer the substance between the first and second states,        a probe detector adapted to detect an optical measurement signal from a portion of the substance in the second state,        
said microscope comprising a phase contrast microscopy system which includes:                the light beam modulating device further adapted to generate a phase contrast,        an illuminating light source arranged to emit an illuminating light beam towards the sample, said illuminating light beam having an intensity and a phase profile,        an intensity detector arranged to detect the intensity of the illuminating light beam after said illuminating light beam has passed at least once through the sample, the objective lens assembly and the light beam modulating device.        